Phaffia rhodozyma is a reddish yeast species producing astaxanthin, a useful natural pigment. Astaxanthin is a member of the carotenoids, which are represented by β-carotene, a precursor of vitamin A. Astaxanthin as a main pigment of curstacea, trout and salmon is widely distributed in nature. However, they cannot synthesize astaxanthin and should be supplied with it from a diet. Thus, it has been considered necessary to add the pigment in the cultivation of crustacea, trout and salmon, because the added pigments to the crustacea and fishes may attract the consumers and give better flavors to them. This carotenoid pigment plays key roles in the physiological metabolism of human as well as animals, with known effects such an enhancement of immunological function, an antioxidant activity, a prevention of cancer and senescence, etc.
Because of increasing interests in Phaffia rhodozyma and pigments produced thereby, there have been a number of reports concerning a culture of Phaffia rhodozyma. However, these reports have been focused on how the inexpensive materials can be used for its culture, and have resulted in the development of method for culturing Phaffica rhodozyma, in which various local products may be employed, such as alfalfa juice (Okagbue et al., Appl. Microbiol. Biotechnol., 20, 33, 1984), molasses (Haard et al., Biotechnol. Lett., 10, 609, 1988), the byproducts of grape juice processing (Lango et al., Biotech. Forum Europe, 9, 565, 1992), peat hydrolyzate (Martin et al., 58, 223, 1993), the byproducts of corn wet-milling (Hayman et al., J. Ind. Microbiol., 14, 389, 1995), and the mixture of sugar cane extract, urea and phosphoric acid (Fontana, et al., Appl. Biochem. Biotechnol., 57/58, 413, 1996).
Although little is known about the genetics of Phaffia rhodozyma, the physiological features of Phaffia rhodozyma have been disclosed and the Phaffia rhodozyma mutant producing the pigment with high level has recently been selected (Johnson et al., Crit. Rev. Biotechnol., 11, 297, 1991; An et al., Appl. Environ. Microbial., 55, 116, 1989; Chumpolkulwong et al., J. Ferment. Bioeng., 75, 375, 1997; Lewis et al., Appl. Environ. Microbiol., 56, 2944, 1990). In addition, a genetic analysis enlightened the ploidy and sexual cycle of Phaffia rhodozyma. In a flow cytometry study, Calo-Mata and Johnson found that no strain was haploid and that most were polyploid (Calo-Mata et al., Yeast Gen. Mol. Biol. Meet., 126, 1996). A pedogamic sexual process of conjugation has been also desclosed (Golubev et al., Yeast, 11, 101, 1995).
Although Phaffia rhodozyma is potentially useful for the production of astaxanthin and the like, the pigment level in the wild type of Phaffia rhodozyma is very low. Therefore, there have been attempts to develop a novel mutant strain of Phaffia rhodozyma, which can produce the pigment more than usual one. However, these attempts have been hampered by the reduced growth rate and genetic instability of said mutant, which may occur when the pigment content in the mutant exceeds over the optimal range.
Another obstacle to the progress of the mutant is the method for mutagenesis. Chemical mutagenesis procedures have been performed conventionally, but it is associated with the simultaneous mutation of undesired genes leading to pleiotropic effects such as the reduction of growth rate, the prolonged induction time in the fermentation, etc. Furthermore, because the genome of the mutant strain is not stable, its subculture often decreases the yield of the pigment.
To solve these problems in the conventional breeding procedures and to enlarge the applicability of Phaffia rhodozyma, molecular breeding approaches have been initiated recently, using genetic transformation. However, since most of Phaffia rhodozyma strains are polyploid and thus cannot be made to be an auxotrophic variant by the method conventionally applied to yeast, it is preferable to employ an approach using antibiotics-resistant genes as selectable markers. More recently, there was reported a transformation system in which Phaffia rhodozyma actin promoter and G418-resistant gene were used for the transformation of Phaffia rhodozyma, However, the system showed poor transformation efficiency (Wery et al., Gene, 184, 89, 1997).
On the other hand, cycloheximide, an eukaryote-specific antibiotics, is applicable to the selection of yeast transformants. The target molecule of cycloheximide is ribosome and its target site is aminoacyl-tRNA binding site (A site) of ribosome, wherein it blocks peptidyl transferase activity of ribosome. As a result, it inhibits protein synthesis and cell growth in eukaryotes, without an effect on the organelles such as chloroplasts and mitochondria. Furthermore, it has been found that cycloheximide interacts with ribosomal protein L41, and that a mutation in L41 gene confers cycloheximide-resistance on the yeast transformants. Thus, cycloheximide and related mutant form of L41 gene are widely applicable to the process for transformation of yeasts.
Recent studies support the applicability of L41 gene to selectable marker in yeasts. Takagi et al. found that amino acid substitution through the mutagenesis of Saccharomyces cerevisiae L41 gene conferred cycloheximide-resistance, suggesting the usefulness of L41 gene as a selectable marker (Takagi et al., J. Bacteriol., 174, 254–262, 1992). Mutoh et al. proposed a biotechnological tool using Candida maltosa L41 gene as a selectable marker (Mutoh et al., J. Bacteriol., 5383, 177, 1995). As it is well known that a substitution of 56th amino acid residue in the L41 protein conffer cycloheximide-resistant on Candida utilis (Keiji Kondo et al., J. Bacteriol., 7171, 177, 1995), transformation system using the substitution has been developed. Similar approaches have been introduced in Kluyveromyces lactis and Schwanniomyces occidentalis (Dehoux et al., Eur. J. Biochem., 213, 841–843, 1993; Pozo et al., Eur. J. Biochem., 213, 849–857, 1993). On algae Tetrahymena, the resistance is conferred by substitution of 40th amino acid residue, methionine to glutamine (Roberts et al., Exp. Cell. Res., 312, 81, 1973).
To overcome the foregoing and other disadvantages, we, the inventors of the present invention, have noted that cycloheximide and related mutation in L41 gene may be used to develop an efficient transformation system, whereby a foreign gene is stably integrated into the genome of Phaffia rhodozyma, and the transformants are undoubtedly selected. To develop such system, we have constructed transforming vectors comprising the antibiotics-resistant gene and the targeting gene, which is used for the stable integration of foreign gene. We transformed Phaffia rhodozyma with asid vectors, according to a modified method for electrotransforming Cryptococcus neoformans, a member of Basidiomycetes, whereto Phaffia rhodozyma belongs (Kim et al., Appl. Environ. Microbiol., 64, 1947, 1998).
The present invention is performed by cloning and sequencing Phaffia rhodozyma L41 gene; modifying the L41 gene by the mutagenesis of the region responsible to cycloheximide-resistance; constructing the vectors for transforming by inserting ribosomal DNA into the modified L41 gene; transforming Phaffia rhodozyma with the vector by electroporation method; and verifying the stable integration of the vector into the genome of the transformants.